Instrument and method for automatically heat-sealing a microplate

ABSTRACT

A heating device for heating a thermally fixable sealing cover disposed over the microplate adjacent the wells, a cooling device for actively cooling the microplate and a controller set up to control activity of the heating and cooling devices in a manner to heat the sealing cover so as to thermally fix it to the microplate and to actively cool the microplate so as to keep a temperature of the samples below a predefined temperature when heating the sealing cover. It further relates to a method for automatically sealing a microplate in which the thermally fusible sealing cover is disposed over the microplate, the sealing cover is heated to thermally fix it to the microplate and the microplate is actively cooled in a manner that a temperature of the liquid reaction mixtures is kept below a predefined temperature when heating the sealing cover.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C. §119 of EP10189640.5, filed Nov. 2, 2010, the contents of which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention is in the field of clinical analysis and medicaldiagnostics and more particularly relates to an instrument and methodfor automatically heat-sealing a microplate. It further pertains to asystem for thermally processing liquid samples such as reactionmixtures.

BACKGROUND OF THE INVENTION

In these days, nucleic acids (DNA=deoxyribonucleic acid, RNA=ribonucleicacid) are subject to various analyses and assays in clinical analysisand medical diagnostics. Since the initial amount of nucleic acidsnormally is very low, nucleic acids have to be amplified prior to theiruse so as to obtain sufficient amounts which can be used as startingmaterial.

The amplification of nucleic acids using the well-known polymerase chainreaction (PCR) has been extensively described in the patent literature,for instance, in U.S. Pat. Nos. 468,203, 4,683,195, 4,800,159 and4,965,188. Generally, in the polymerase chain reaction, samplescontaining reaction mixtures of specific reagents and nucleic acids arerepeatedly put through a sequence of amplification steps. Each sequenceincludes a step of melting the double-stranded nucleic acids to obtaindenaturated single polynucleotide strands, a step of annealing shortprimers to the strands and a step of extending those primers tosynthesize new polynucleotide strands along the denaturated strands tomake new copies of double-stranded nucleic acids. Due to the fact thatreaction conditions strongly vary with temperatures, the samples are putthrough a series of temperature excursions in which predeterminedtemperatures are kept constant for specific time intervals(“thermo-cycling”). The temperature of the samples typically is raisedto around 90° C. for melting the nucleic acids and lowered to atemperature in the range of from 40° C. to 70° C. for annealing andprimer extension along the polynucleotide strands. It is known to detectthe reaction products even during the progress of the polymerase chainreaction (“real-time PCR”) to thereby obtain more information about theamplification process and to improve the reliability of the detectionresults.

In daily routine, the PCR is performed in commercially availableinstruments enabling a large number of reaction mixtures to be cycledsimultaneously. Usually, integrally molded plastic disposables providedwith plural open-top wells sized to receive the reaction mixtures areused for nucleic acid amplification. Such disposables are commonly knownas “microplates”.

It has been found advantageous to provide the open-top wells with asealing cover for air-tightly sealing individual wells containing thereaction mixtures. One reason is the necessity to avoid evaporation ofliquids so as to ensure the integrity of the reaction mixtures. Anotherreason is to prevent spilling of the contents of the wells duringtransport of the microplate from one location to another. A yet anotherreason is to prevent cross contamination of individual reaction mixturescontained in the wells so as to provide a generally sterile andcontrolled environment under which the amplification steps can becarried out.

It is convenient to use transparent sealing covers such as thin plasticfoils applied to the top surface of the microplate which allow for anoptical detection of the reaction products even during progress of thereactions. In practical use, for instance, an adhesive plastic foilprovided with an adhesive backing is positioned over the microplate sothat the adhesive backing faces the upper surface of the microplate. Theplastic foil then is pressed on the upper surface, e.g., by means of apressure roll rolling back and forth to thereby obtain uniform adhesionof the sealing foil to the microplate. Adhesive foils, however, oftencause problems with respect to an air-tight sealing of individual wells.Accordingly, undesired evaporation of fluids impairing thereproducibility of test results especially in the case of small samplevolumes and cross contamination between various reaction mixtures mayoccur. Otherwise, the adhesive material may probably influence theoutcome of the nucleic acid amplification steps thus downgrading thereliability of the test results.

Better results can normally be obtained using thermally fusible foils.In practical use, the foil is positioned over the microplate and heated,e.g., by means of a heated sealing stamp which can be brought in and outof contact with the foil. While heated, the sealing foil is pressed ontothe microplate to ensure a close adhesive fit with full contact to themicroplate.

In light of the foregoing, it is an object of the invention to providean improved instrument and method for automatically heat-sealingmicroplates. It is a further object of the invention to provide animproved system for processing, especially thermally processing, and/oranalyzing liquid samples. These and further objects are met by aninstrument and method for thermally heat-sealing microplates as well asa system for thermally processing liquid samples according to theindependent claims. Preferred embodiments of the invention are given bythe features of the dependent claims.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, a new instrument for theautomated heat-sealing of a microplate using a thermally fixable sealingcover is proposed. In some embodiments, the microplate has an uppersurface and an opposing lower surface wherein a plurality of open-topretention regions or wells for receiving liquid samples such as reactionmixtures is formed in the upper surface.

In some embodiments, the instrument of the invention comprises a heatingdevice for heating the thermally fixable sealing cover disposed over themicroplate adjacent the wells. In some embodiments, the thermallyfixable sealing cover is a thermally fusible sealing cover. In somealternative embodiments, the sealing cover is provided with an adhesivematerial which can be thermally activated. Such adhesive materials arewell-known to those of skill in the art and, e.g., are described in U.S.Pat. No. 7,037,580. In some embodiments, the heating cover is disposedon the upper plate surface of the microplate. Heated by the heatingdevice, in some embodiments, the sealing cover can at least locally beheated, e.g., fused so as to induce adherence to the microplate, e.g.,going along with solidification of the sealing cover in order toair-tightly seal each of the wells. For heating the sealing cover, theheating device can be brought in and out of thermal communication withthe sealing cover. In some embodiments the heating device can also bebrought in and out of direct (physical) contact with the sealing cover,especially, in order to press the sealing cover on the microplate. Insome embodiments, the sealing cover is configured as transparent sealingcover enabling optical detection of reaction products contained in thewells. In some embodiments, the heating device is adapted to be operatedto heat or alternatively actively cool the sealing cover. In the lattercase, the heating device can, e.g., include one or more thermoelectricdevices (Peltier devices) which can generate or absorb heat according tothe direction of the current applied. Accordingly, solidification of thesealing cover can advantageously be accelerated.

In some embodiments, the instrument of the invention comprises a coolingdevice for actively cooling the microplate. In some embodiments, thecooling device comprises a casing forming an internal space which can beactively cooled. The internal space preferably accommodates the heatingdevice for thermally fixing the sealing cover to the microplate in anactively cooled environment. It is preferred to accommodate a heatingdevice having a low thermal capacity in the internal space. In someembodiments, the casing is a closed casing. As used herein, the term“closed casing” relates to a casing which contrary to the strict senseof the term can be provided with one or more openings or ports, e.g., totransfer microplates into and out of the internal space. In someembodiments, the casing is provided with one or more ports which areconfigured to be brought in a temporarily opened or closed state.Generally, the closed casing which can be provided with one or moreports serves to restrict or reduce the input of heat and liquid fluidsto the internal space so as to provide a controlled environment in orderto prevent contamination of liquid samples contained therein. In someembodiments, the closed casing is provided with at least one microplateport which can be closed or opened for transporting the microplate in orout of the internal space. The internal space can be actively cooled bya cooling means in thermal communication with the internal space. Insome embodiments the cooling means is a cooling coil adapted forcirculating cooling fluid. In some other embodiments, one or morethermoelectric devices based on the Peltier effect are used for coolingthe internal space. As is known to the skilled persons, when passingelectric current through a Peltier device, depending on the direction ofcurrent applied, the Peltier device functions as heat sink which absorbsheat or as heat source which releases heat to thereby cool or heat theinternal space. In some embodiments, the cooling device is integrated inthe heating device, that is to say, the heating and cooling device has acooling and heating function. In this case, the heating and coolingdevices, e.g., are configured as one or more thermoelectric devices forboth heating and cooling the liquid samples. In some embodiments, theinstrument of the invention comprises a controller set up to controlactivity of each of the heating and cooling devices in a manner to heatthe sealing cover so as to thermally fix it to the microplate and toactively cool the microplate so as to keep the temperature of the liquidsamples contained in the wells below a predefined temperature.

In some embodiments related to the PCR, the microplate is activelycooled in a manner to keep the temperature of the liquid samplescontained in the wells below a predefined critical temperature lowerthan the temperature excursions of the nucleic acid amplification stepswhen heating the sealing cover. The predefined critical temperature isconsidered critical for starting the nucleic acid amplification. Inother words, the predefined critical temperature is selectively chosento prevent the start of any unspecific (inadvertent) nucleic acidamplification caused by heat-sealing the microplate prior to thermallycycling the reaction mixtures. In some embodiments, the predefinedcritical temperature is chosen to be 40° C. so that the temperature ofthe liquid reaction mixtures is always below 40° C. during theheat-sealing process. In some embodiments the predefined criticaltemperature is selected from a temperature range of from 32° C. to 40°C. In the latter case, the predefined critical temperature may, e.g., beselected from the group of temperatures consisting of 39° C., 38° C.,37° C., 36° C., 35° C., 34° C., 33° C. and 32° C. Otherwise, in someembodiments, dependent on the temperature of the reaction mixtures priorto starting the heat-sealing, it can be preferred that a temperatureraise of the liquid reaction mixtures caused by heat-sealing themicroplate is lower than 4° C. In some embodiments it may be preferredthat the temperature raise is lower than 3° C., more preferably lowerthan 2° C. or even more preferably lower than 1° C. The instrument ofthe invention thus enables a precise temperature control of the reactionmixtures preventing any unspecific nucleic acid amplification prior tothermal cycling so as to improve the reliability and reproducibility ofthe nucleic acid amplification.

In some embodiments, the controller is set up to actively cool themicroplate simultaneously with heating the sealing cover. In someembodiments, the controller is set up to actively cool the microplateprior to heating the sealing cover. In some embodiments, the controlleris set up to actively cool the microplate prior to and simultaneouslywith heating the sealing cover.

In some embodiments, the instrument of the invention comprises a baseand a base-mounted holder adapted for holding the microplate. In someembodiments, the holder is configured as tray slidably mounted to thebase for a repetitive, bidirectional movement between an operativeposition for thermally fixing the sealing cover to the microplate and aninoperative position for loading or unloading the microplate to/from theholding tray. In some embodiments, the sealing arrangement includes amoving mechanism for moving the holding tray between its operative andinoperative positions. In some other embodiments, the holding tray canbe manually moved between its operative and inoperative positions. Insome embodiments comprising a casing forming an actively cooled internalspace as above-detailed, the inoperative position is located outside thecasing. Due to the microplate port which can be opened or closed, aconsiderable increase of the temperature of the actively cooled internalspace of the casing an advantageously be prevented.

In some embodiments, the heating device comprises a supporting layerprovided with at least one electrically conductive heating elementadapted for generating Ohmic heat. In some embodiments, the supportinglayer is made of an electrically isolating material. In someembodiments, the heating element is applied to an outer surface of thesupporting layer. In some embodiments, the heating element is a meshedstructure consisting of electrically conductive lines which, e.g., canbe configured as conductive wires.

In some embodiments, the supporting layer is made of material having lowthermal capacity such as ceramic material or plastic material likepolyimide. Accordingly, the heating device can quickly be heated andcooled enabling a time- and cost-efficient sealing of the microplate.Otherwise, a major advantage is given by the fact that only little heatis lost in the instrument thus improving power efficiency in operatingthe instrument. Another advantage of a heating device having low thermalcapacity is given by the fact that it can be located inside the casingof a cooling device forming an internal space which can be activelycooled.

In some embodiments, especially in case of using materials having lowthermal capacity, the supporting layer can, e.g., have a layer thicknessof less than 3 mm so as to reduce the total energy up-take of thesupporting layer. It can be preferred that the supporting layer has alayer thickness of, e.g., 1.0 mm or 0.5 mm or less.

In some embodiments, the heating device is provided with a plurality ofnon-heated zones arranged in opposite relationship with respect toopenings of the wells when the heating device is in thermalcommunication and eventually in physical contact with the sealing cover.Accordingly, the sealing cover can be heated exclusively in regionswhere adhesion to the microplate is to be reached so as to reduce theheat load of the microplate and to reduce the temperature raise of themicroplate. In some embodiments, the non-heated zones are configured bygaps between the electrically conductive wires of the meshed heatingelement.

In some embodiments the heating device comprises one or more Peltierelements to generate the heat for sealing. When using a Peltier elementthe same element can also be used to cool the sealing zone in order toaccelerate solidification of the sealing foil.

In some embodiments the heating device is adapted to generate the heatfor sealing the sealing cover onto the microplate by inductive heating.

In some embodiments, the heating device includes a rigid pressing layerfor pressing the sealing cover on the microplate. In some embodiments,the pressing layer is fixed to the supporting layer in oppositerelationship with respect to the heating element. In some embodiments,the heating element includes an isolating layer made of thermallyisolating material such as polytetrafluoroethylene (PTFE) sandwichedin-between the supporting and pressing layers so as to inhibit heattransfer from the supporting layer to the pressing layer. Accordingly,heat uptake of the pressing layer and heat loss can advantageously befurther reduced so as to improve power efficiency in operating theinstrument.

In some embodiments, the holder for holding the microplate is providedwith one or more resilient elements such as compression springs orelastic gum counteracting the pressing force of the pressing layer inorder to obtain a homogenous contact pressure and to level themicroplate. Otherwise, a full contact of the sealing cover with closefit to the microplate even in case of a slightly non-planar microplatecan advantageously be obtained. In some embodiments, the microplate ismade of plastic material having a plate height of a few millimeters sothat the microplate has sufficient flexibility to be planarized underaction of the pressing layer. In some embodiments, the rigid pressinglayer is additionally and/or alternatively being used for levelling themicroplate.

According to a second aspect of the invention, a new system forprocessing, in particular thermally processing (e.g. incubating) and/oranalyzing liquid samples such as reaction mixtures is proposed. Thesystem of the invention can be configured in various ways in accordancewith specific demands of the user. Stated more particularly, the systemof the invention can be used for processing liquid samples whereinprocessing of the liquid samples involves pipetting of the samples bymeans of one or more pipettors. Accordingly, in some embodiments, thesamples are subject to pipetting operations prior to heat-sealing thewells of the microplate, e.g., to pipette the samples into the wellsand/or to add fluids to and/or to remove aliquots from the samplescontained in the wells. In some embodiments, the samples contained inthe wells are subject to pipetting operations after heat-sealing thewells, e.g., to add fluids to the samples contained in the wells and/orto remove aliquots therefrom. Additionally or alternatively, the systemof the invention can be used for analyzing liquid samples contained inthe heat-sealed wells by means of one or more analytical compartmentsfor performing tests and assays related to various immunochemical and/orclinical-chemical analysis items.

In some embodiments, the system is adapted to thermally process reactionmixtures contained in the heat-sealed wells to be put through a seriesof temperature excursions, e.g., for performing the PCR or any otherreaction of the nucleic acid amplification type. Specifically,transparent sealing covers enable quantitative real-time PCR byoptically detecting the reaction products obtained during progress ofthe reactions. Reaction mixtures for thermal processing by the system ofthe invention typically include biological material containing nucleicacids but may also contain any other substance of interest as long asthe processing thereof requires thermal treating. According to theinvention, the system is equipped with at least one instrument forautomatically heat-sealing a microplate as above-detailed.

According to a third aspect of the invention, a new method forautomatically heat-sealing a microplate provided with a plurality ofopen-top wells for receiving liquid samples such as reaction mixturesfor thermally cycling through a series of temperature excursions isproposed. The method of the invention comprises a step of disposing athermally fixable sealing cover over the microplate. In someembodiments, the thermally fixable sealing cover is a thermally fusiblesealing cover. In some embodiments, the thermally fixable sealing coveris provided with an adhesive material which can be thermally activated.The method comprises a further step of heating the sealing cover so asto fix the sealing cover to the microplate and a yet further step ofactively cooling the microplate so that the temperature of the liquidreaction mixtures contained in the wells is kept below a predefinedtemperature when heating the sealing cover. In some embodiments, whenheating the sealing cover, the temperature of the liquid reactionmixtures contained in the wells is kept below a predefined criticaltemperature lower than temperature excursions when performing the PCR.In some embodiments, the microplate is actively cooled simultaneouslywith heating the sealing cover. In some embodiments, the microplate isactively cooled prior to heating the sealing cover. In some embodiments,the microplate is actively cooled prior to and simultaneously withheating the sealing cover.

In some embodiments, the sealing cover is exclusively heated in regionsin opposite relationship with respect to protruding rims surroundingopenings of the wells. Accordingly, the sealing cover is not heated inregions other than those regions. In some embodiments, the sealing coveris exclusively heated in regions located in-between adjacent openings ofthe wells. Accordingly, the sealing cover can exclusively be heated inselected regions where adhesion to the microplate is to be reached onlyso as to reduce the total heat input to the microplate.

In some embodiments, the sealing cover is pressed on the microplatewhile the sealing cover is heated. In some embodiments, the pressingaction ends after stopping heating the sealing cover. In someembodiments, it can be preferred that the pressing action ends afterhaving the sealing cover solidified. Accordingly, in some embodimentsinvolving the use of a thermally fusible sealing cover, the pressingaction can continue until a solidified state of the sealing cover isreached advantageously enabling an easy and cost-efficient constructionof the sealing cover.

In some embodiments, the microplate is cooled prior to starting heatingthe sealing cover so as to start heat-sealing of the microplate with apre-cooled microplate containing pre-cooled reagent mixtures. In someembodiments, the microplate is cooled after heat-sealing so as to coolthe reagent mixtures contained in the wells prior to thermal cycling.Accordingly, the sealing arrangement can advantageously be used as coolstorage for storing the microplate before and/or after the heat sealing.

BRIEF DESCRIPTION OF THE FIGURES

Other and further objects, features and advantages of the invention willappear more fully from the following description. The accompanyingdrawings, together with the general description given above and thedetailed description given below, serve to explain the principles of theinvention.

FIG. 1 shows a schematic sectional view of an exemplary instrument ofthe invention;

FIG. 2 shows a schematic perspective view of an exemplary system of theinvention.

FIG. 3 shows a process scheme of sealing a sealing cover to a microplate

DETAILED DESCRIPTION OF THE INVENTION

By way of illustration, specific exemplary embodiments in which theinvention may be practiced now are described. In this regard,terminology with respect to orientations and directions such as“horizontal”, “vertical”, “upper”, “lower” is used with reference to theorientation of the figure being described. Because components describedcan be positioned in a number of different orientations, thisterminology is used for the purpose of illustration only and is in noway limiting.

First, reference is made to FIG. 1. Accordingly, in some embodiments, aninstrument generally referred to at reference numeral 1 for heat-sealinga microplate 3, includes a cooling device 19 comprising a closedinstrument casing 6 forming an internal instrument space 10 which can beactively cooled. As detailed in the introductory portion, the term“closed” relates to a casing 6 which can be provided with openings orports. With continued reference to FIG. 1, in some embodiments, theinternal instrument space 10 can be cooled by means of a cooling coil 8.The cooling coil 8 penetrates the instrument casing 6. On its ends it isprovided with fluid ports 9 for supplying cooling fluid such as water orair for circulating through the cooling coil 8. It is to be appreciatedthat any other technique for cooling the internal instrument space 10can also be used. Specifically, in some embodiments, cooling of theinternal space 10 can alternatively be based on thermoelectric devicesusing the Peltier effect.

With continued reference to FIG. 1, in some embodiments, the instrument1 includes a tray 11 supporting the microplate 3 in horizontal position.In some embodiments, the tray 11 is slidably mounted to a base 12enabling a repetitive, bidirectional movement between an operative orsealing position inside the instrument casing 6 for heat-sealing themicroplate 3 and an inoperative or loading/unloading position outsidethe instrument casing 6 for loading/unloading the microplate 3 on/fromthe tray 11. As schematically illustrated in FIG. 1, in someembodiments, the instrument casing 6 is provided with a microplate port7 so that the tray 11 can be horizontally moved through the microplateport 7. The microplate port 7 can be closed or opened by a closing meanssuch as a door (not illustrated) so as to enable transport of the tray11 with or without microplate 3 through the microplate port 7. Sincesuch sliding mechanism is well-known to those of skill in the art, itneed not be further elucidated herein. In some embodiments, theinstrument 1 includes a moving mechanism for automatically moving thetray 11 between its sealing and loading/unloading positions. Since suchmoving mechanism is also well-known to those of skill in the art, itneed not be further elucidated herein.

In some embodiments, an upper plate surface 4 of the microplate 3 formsa plurality of open-top wells 5 for receiving liquid samples such asreaction mixtures for performing the PCR which typically includebiological material containing nucleic acids. With continued referenceto FIG. 1, in some embodiments, the wells 5 are regularly arranged in atwo-dimensional array of, e.g., ninety-six wells 5 comprised of eightcolumns and twelve rows intersecting each other at right angles. It,however, is to be appreciated that any other number of wells 5 may beenvisaged according to the specific demands of the user. In someembodiments, the microplate 3 is an integrally moulded plasticdisposable intended for single use only.

Further referring to FIG. 1, in some embodiments, the tray 11 isprovided with a plurality of helical compression springs 14. Asillustrated, in some embodiments, the compression springs 14 arearranged in correspondence to the wells 5 of the microplate 3 whereuponthe number of compression springs 14 may, e.g., correspond to the numberof the wells 5. Each of the compression springs 14 can, e.g., be adaptedto accommodate one well 5 in a close fit. With continued reference toFIG. 1, in some embodiments, the compression springs 14 are in anupright position relative to an upper tray surface 13 in parallelalignment with respect to each other. Having their lower and upper ends16, 17 in contact with the tray 11 and microplate 3, respectively, thehelical compression springs 14 can elastically be compressed between thetray 11 and the microplate 3. Grace to a non-zero freeboard 18 betweenthe wells 5 and the upper tray surface 13, the microplate 3 canvertically be moved against the elastic force of the compression springs14. Otherwise, by effect of inserting the wells 5 into the compressionsprings 14, the microplate 3 is horizontally secured by the compressionsprings 14. In some embodiments, the instrument 1 is equipped with oneor more resilient means other than compression springs 14 such as anelastic gum or rubber for elastically holding the microplate 3.

In some embodiments, the instrument 1 further includes a heating device20 which, with continued reference to FIG. 1, in some embodiments,comprises a thin supporting layer 21, e.g., made of electricallyisolating material having a low thermal capacity such as a ceramic orplastic material. The supporting layer 21 can, e.g., be made ofpolyimide having a layer thickness as small as 0.5 mm.

With continued reference to FIG. 1, in some embodiments, a heatingelement 26 adapted for generating Ohmic heat is fixed to a lower layersurface 22 of the supporting layer 21. In some embodiments, the heatingelement 26 includes a plurality of electrically conductive heating lines27 connected to a connecting line 31 for supplying electric current tothe heating element 26. In some embodiments, the heating lines 27 areembedded in a carrier layer (not illustrated) made of isolating materialsuch as plastic enabling the heating element 26 to be readily fixed tothe supporting layer 21.

As illustrated in FIG. 1, in some embodiments, an isolating layer 24made of isolating material is fixed to an upper layer surface 23 of thesupporting layer 21. The isolating layer 24 can, e.g., be made ofpolytetrafluoroethylene (PTFE) commonly known as Teflon and have a layerthickness in the range of from 3 to 5 mm. Those of skill in the art willappreciate that any other material and/or layer thickness can beenvisaged according to the specific demands of the user.

With continued reference to FIG. 1, in some embodiments, a rigidpressing layer 25 is fixed to the upper side of the isolating layer 24.The pressing layer 25 can, e.g., be made of aluminium and have a layerthickness of 10 mm. However, other rigid materials and/or other layerthicknesses can be envisaged according to the specific demands of theuser. Sandwiched in-between the supporting and pressing layers 21, 25,the isolating layer 24 inhibits heat transfer from the supporting layer21 to the pressing layer 25.

As schematically illustrated in FIG. 1, in some embodiments, the heatingdevice 20 can be vertically moved so as to bring the heating element 26in and out of physical contact with a thermally fusible sealing cover 30located over the microplate 3 adjacent the wells 5. In some embodiments,the sealing cover 30 is placed on the upper plate surface 4 of themicroplate 3. Since such moving mechanism is well-known to those ofskill in the art, it need not be further elucidated herein. Due to therigid pressing layer 25 backing the supporting layer 21, the sealingcover 30 can be pressed on the microplate 3 while heated by the heatingelement 26 acting against the elastic forces of the helical compressionsprings 14. Due to the resilient forces of the helical compressionsprings 14, a homogeneous pressure force can act on the microplate 3 forlevelling the microplate 3 at a pre-defined height. Otherwise, underaction of the heating device 20, the microplate 3 can be presseddownwards using the freeboard 18 to thereby ensure a close fit of thesealing cover 30 even in case of a slightly non-even microplate 3 whichcan be planarized.

With continued reference to FIG. 1, in some embodiments, the heatinglines 27 form a mesh-like wired structure adapted to contact the sealingcover 30 exclusively in regions where the sealing cover 30 is inopposite relationship to rims 29 projecting from the upper plate surface4. Each of rims 29 surrounds an opening 32 of one well 5. Accordingly,the sealing cover 30 can exclusively be heated, e.g., fused at the rims29. Otherwise, as illustrated in FIG. 1, non-heated zones 28 between theheating lines 27 are in opposite relationship with respect to theopenings 32 of the wells 5 when the heating element 26 contacts thesealing cover 30 for heat-sealing.

In some embodiments, as illustrated in FIG. 1, the instrument 1 furtherincludes an instrument controller 2 set up to control heat-sealing ofthe microplate 3. The instrument controller 2 can, e.g., be embodied asprogrammable logic controller running a computer-readable program. Theinstrument controller 2 is electrically connected to the instrumentcomponents which require control and/or provide information whichinclude the cooling device 19 and the heating device 20.

In practical use, under control of the instrument controller 2, in someembodiments, the tray 11 is horizontally moved through the microplateport 7 into inoperative position outside the instrument casing 6 wherethe microplate 3 containing the liquid samples such as reaction mixturescan be put on the tray 11. The tray 11, together with the microplate 3,is then horizontally moved into operative position inside the instrumentcasing 6 where the microplate 3 is kept ready for heat-sealing.

In some embodiments, the thermally fixable sealing cover 30 is placedover the microplate 3 in sealing position. In some other embodiments,the sealing cover 30 is placed over the microplate 3 prior totransporting the microplate 3 into sealing position, particularly in asituation where the microplate 3 is located outside the instrumentcasing 6.

In some embodiments, the heating device 20 is vertically moved until theheating element 26 is in thermal communication with the sealing cover 30so as to heat, e.g., thermally fuse the sealing cover 30 in regionsopposing the rims 29. With reference to FIG. 1, is can be preferred thatthe heating device 20 is vertically moved until the heating element 26is in (physical) contact with the sealing cover 30. In some embodiments,the heating device 20 is pressed on the microplate 3 simultaneously withheating the sealing cover 30.

In some embodiments, the microplate 3 is actively cooled prior andsimultaneously with heating the sealing cover 30 so as to prevent theliquid samples from experiencing an undesired high temperature increaseso that, e.g., in the case of performing the PCR, the temperature of thereaction mixtures is below a pre-defined critical temperature. In someembodiments, the predefined temperature can be 40° C. which cansometimes be considered critical for initiating unspecific nucleic acidamplification. In some embodiments, e.g., starting with reactionmixtures having a temperature of about 32° C., a temperature increase ofthe reaction mixtures contained in the wells 5 is below 5° C.

Heating of the sealing cover 30 then is stopped, e.g., to have thesealing cover 30 solidified so as to cause adherence of the sealingcover 30 to the microplate 3 in order to air-tightly seal the wells 5.In some embodiments, the pressing action of the heating device 20 isstopped not before the sealing cover 30 is in a fully solidified state.

In some embodiments the sealing zone is actively cooled to acceleratesolidification of the sealing cover 30 (sealing foil) and/or moltenportions of the microplate 3. Such active cooling can e.g. be done by afan (not illustrated). Those of skill in the art will appreciate thatany other means for actively cooling the sealing cover 30 accommodatedin the instrument space 10 of the instrument 1 can be envisagedaccording to the specific demands of the user. Specifically, the heatingdevice 20 can for instance include a plurality of thermoelectric devices(Peltier devices) which based on the Peltier effect and depending on thedirection of the current applied can be operated to alternatively heator cool the sealing cover 30.

The heating device 20 is then moved upwards, followed by moving the tray11 into the inoperative position outside the instrument casing 6 so thatthe microplate 3 can be removed from the tray 11 for further processing,i.e. thermal cycling, of the reaction mixtures contained therein.

Accordingly, in the case of performing the PCR, unspecific amplificationof reaction mixtures contained in the wells 5 caused by heat-sealing themicroplate 3 can advantageously be avoided. Due to the combined measuresthat

-   -   the sealing cover 30 is exclusively heated in regions opposing        the rims 29,    -   the sealing cover 30 is not heated in regions opposing the        openings 32 of the wells 5,    -   the supporting layer 21 (and also the heating element 26) has a        low thermal capacity, and    -   heat transfer to the pressing layer 25 is strongly inhibited by        the isolating layer 24,        various synergistic positive effects, e.g., with respect to a        low heat input into the instrument casing 6 for heat-sealing the        microplate 3 and low heat loss inside the instrument casing 6        during and following the heat-sealing process in the cooling-off        period of the heating device 20 and thereby an improved power        efficiency can be obtained. Otherwise, the heating element 26        can quickly be heated and cooled to thereby reduce the time        interval needed for heat-sealing the microplate 3. In the case        of using a thermally fusible sealing cover 30, due to the quick        cooling, another major advantage is given by the fact that        pressing of the sealing cover 30 to the microplate 3 can also be        stopped after having the sealing cover 30 fully solidified thus        enabling a simple and cost-effective structure of the sealing        cover 30. Stated more particularly, in contrast to the        conventional construction, the sealing cover 30 may not        necessarily have a laminated structure consisting of several        individual layers such as a fusible layer, adhesive layer and        separating layer so as to not cause a poorly adhering or even        damaged sealing cover when removing the heating element 26.        Accordingly, the sealing cover 30 can be produced in a highly        cost-effective manner. Otherwise, optical transparency of the        sealing cover 30 can be improved. Solidification of the sealing        cover 30 can also be accelerated by actively cooling the sealing        cover 30.

According to the invention, it is highly preferred to provide for aclosed instrument casing 6 in line with understanding of the term“closed” as detailed in the introductory portion the internal instrumentspace 10 of which can be actively cooled, inter alia, for the followingreasons:

-   -   formation of condensate on the microplate 3 is largely reduced,    -   in case of using a thermally fusible sealing cover 30, there is        a quicker solidification of the fused sealing cover 30,    -   the tray 11 can already be in a pre-cooled condition prior to        starting heat-sealing the microplate 3 saving time to heat-seal        the microplate 3.

In some alternative embodiments, not illustrated in FIG. 1, integratedheating and cooling devices 19, 20 can be used, e.g., configured as oneor more thermoelectric devices such as Peltier devices. Depending on thedirection of current applied, the thermoelectric devices canalternatively produce or absorb heat.

Reference is now made to FIG. 2 illustrating a schematic diagram of anautomated system for thermally processing liquid samples. In someembodiments, the system is used for cycling liquid reaction mixturesthrough a series of temperature excursions for performing the PCR or anyother reaction of the nucleic acid amplification type.

As illustrated in FIG. 2, in some embodiments, the system generallyreferred to at reference numeral 33 includes a closed system casing 34(in line with the understanding of the term “closed” as detailed in theintroductory portion) forming an internal system space 35, inter alia,containing the instrument 1 for heat-sealing microplates 3 asillustrated in FIG. 1. In some embodiments, the tray 11 is adapted forsupporting the microplate 3 in horizontal position on an upper tray face43. The tray 11 enables a repetitive, bidirectional movement between theinternal system space 35 for loading or unloading the microplate 3on/from the tray 11 and the internal instrument space 10 of theinstrument 1 for heat-sealing the microplate 3 prior to thermallyprocessing the reaction mixtures contained in the wells 5. Since suchsliding mechanism is well-known to those of skill in the art, it neednot be further elucidated herein.

With continued reference to FIG. 2, in some embodiments, the internalsystem space 35 accommodates a temperature-controlled block 36 forheating and/or cooling the liquid reaction mixtures. In someembodiments, the temperature-controlled block 36 contains thermoelectricdevices (not further detailed) using the Peltier effect. On its uppersurface the temperature-controlled block 36 has a generally planar seat38 for accommodating the microplate 3. The seat 38 is provided with aplurality of recesses 39 for receiving the wells 5 of the microplate 3in close fit with at least partially full contact for thermalcommunication between the wells 5 and the temperature-controlled block36.

Accordingly, the reaction mixtures contained in the wells 5 of themicroplate 3 can be thermally cycled through a series of temperatureexcursions. Specifically, in the PCR, a multiply repeated sequence ofsteps for the amplification of nucleic acids is done, wherein in eachsequence the nucleic acids are melted (denaturated) to obtain denaturedpolynucleotide strands, primers are annealed to the denaturatedpolynucleotide strands, and the primers are extended to synthesize newpolynucleotide strands along the denaturated strands to thereby obtainnew copies of double-stranded nucleic acids.

With continued reference to FIG. 2, in some embodiments, the system 33includes a detection arrangement generally referred to at referencenumeral 40 for optically detecting the reaction products of theamplification steps. Stated more particularly, the detection arrangement40 is positioned to detect emission beams emitted from the wells 5 ofthe microplate 3 placed on the seat 38. With yet continued reference toFIG. 2, in some embodiments, the detection arrangement 40 includes oneor more detectors 41 for optically detecting the emitted light such as,but not limited to, charge coupled devices (CCDs), diode arrays,photomultiplier tube arrays, charge injection devices (CIDs), CMOSdetectors and avalanche photo diodes. In some embodiments, the detectionarrangement 40 also includes one or more excitation light sources suchas lamps to excite emission of the emission beams from the reactionproducts. With yet continued reference to FIG. 2, in some embodiments,the detection arrangement 40 further includes light guiding elements 42such as, but not limited to, lenses and mirrors and/or light separatingelements such as, but not limited to, transmission gratings, reflectivegratings and prisms. Specifically, radiation such as excitation lightcan be transmitted to the reaction mixtures and (e.g. fluorescent) lightemitted to the one or more detectors 41 can be detected. Specifically,in the detection arrangement 40, the reaction products can also bedetected during the progress of the reactions commonly known asreal-time PCR. Since the optical detection of reaction products iswell-known to those of skill in the art, it is not further elucidatedherein.

The system 33 further includes a system controller 37 set up to controlthermal processing of the reaction mixtures. In some embodiments, thesystem controller 37 can also be set-up for automatically heat-sealingthe microplate 3 and, thus, can be used instead of the instrumentcontroller 2. The system controller 37 can, e.g., be embodied asprogrammable logic controller running a computer-readable program. Thesystem controller 37 is electrically connected to the system componentswhich require control and/or provide information which include thetemperature-controlled block 36, the detection arrangement 40 and,optionally, the instrument 1.

In practical use, under control of the system controller 37, in someembodiments, the tray 11 loaded with the microplate 3 provided with thereaction mixtures is horizontally moved into the instrument 1 forthermally fixing the sealing cover 30 to the microplate 3. Themicroplate 3 is then placed on the temperature-controlled block 36 forthermal processing of the reaction mixtures, e.g., using the tray 11.Otherwise, in some embodiments, the tray 11 is used for moving themicroplate 3 into and/or out of the internal system space 35.

FIG. 3 shows an exemplary process scheme for sealing the thermallyfixable sealing cover 30 onto the microplate 3. The upper section ofFIG. 3 depicts the temperature of the heated portions of the heatingdevice 20 (“heater”) over time and the lower section thereof illustratesthe vertical position of the heater relative to the sealing cover 30over time. Generally, the sealing process can be divided into severalconsecutive phases, i.e., a preheating phase, a sealing phase and acooling phase.

With continued reference to FIG. 3, prior to the preheating phase, theheater is moved towards the sealing cover 30 and placed thereon so as togenerate mechanical pressure acting on the sealing cover 30 to press thesealing cover 30 onto the microplate 3. Specifically, in the curvedepicted in the lower section of FIG. 3, the ramp increasing over timeindicates the movement of the heater towards the sealing cover 30 tobuild up mechanical pressure acting on the sealing cover 30, theconstant portion thereof indicates a non-varied position of the heaterso as to keep a constant mechanical pressure acting on the sealing cover30, and the ramp decreasing over time indicates the movement of theheater away from the sealing cover 30 to release the mechanical pressureacting on the sealing cover.

During the preheating phase the temperature of the heated portions ofthe heating device 20 is ramped up, as indicated by ramp up phase 50, toa temperature above the melting point of the employed sealing cover 30.During the preheating phase, the heater position is not varied so as tokeep the mechanical pressure of the heater acting against the sealingcover 30 constant.

During the sealing phase the temperature of the heating device 20 isfurther raised to a predetermined level and then is kept constant.During the sealing phase, the heater position is not varied to furtherkeep the mechanical pressure of the heater against the sealing cover 30constant to melt the sealing cover 30 and the microplate 3 together.

The cooling phase is divided in two periods, i.e., a first coolingperiod 51 and a second cooling period 52. During the first coolingperiod 51 the heater position is not varied so as to still keep themechanical pressure acting on the sealing cover 30 constant until thetemperature of the sealing cover 30 falls below the melting temperature53 of the sealing cover 30. Only then, in the second cooling period 52,the heater is moved away from the sealing cover 30 to release themechanical pressure acting on the sealing cover 30. In the coolingphase, the sealing cover 30 can be passively or actively cooled.

With such a sealing process simple one layer sealing cover foils may beemployed. Such foils are on the one hand cheaper and on the other handhave better optical properties than sealing covers where two or morefoils are laminated together.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

REFERENCE LIST

-   1 Instrument-   2 Instrument controller-   3 Microplate-   4 Upper plate surface-   5 Well-   6 Instrument casing-   7 Microplate Port-   8 Cooling coil-   9 Fluid port-   10 Instrument space-   11 Tray-   12 Base-   13 Upper tray surface-   14 Compression spring-   15 Lower plate surface-   16 Lower end-   17 Upper end-   18 Freeboard-   19 Cooling device-   20 Heating device-   21 Supporting layer-   22 Lower layer surface-   23 Upper layer surface-   24 Isolating layer-   25 Pressing layer-   26 Heating element-   27 Heating line-   28 Non-heated zone-   29 Rim-   30 Sealing cover-   31 Connecting line-   32 Opening-   33 System-   34 System casing-   35 System space-   36 Block-   37 System controller-   38 Seat-   39 Recess-   40 Detection arrangement-   41 Detector-   42 Light guiding element-   43 Upper tray face-   50 Ramp up phase-   51 First cooling period-   52 Second cooling period-   53 Melting temperature

What is claimed:
 1. A method for automatically heat-sealing a microplateprovided with a plurality of open-top wells surrounded by protrudingrims, said wells being configured for receiving liquid samples, saidmethod comprising the following steps: disposing a thermally fixablesealing cover over said microplate, wherein said sealing cover comprisesregions in opposite relationship with respect to said protruding rims;contacting said regions of said sealing cover with a heating devicecomprising (a) a plurality of discrete electrically conductive heatingelements each individually configured to generate Ohmic heat, and (b) aplurality of non-heated zones positioned adjacent to the heatingelements and in opposite relationship with respect to the open-topwells; exclusively heating the plurality of discrete electricallyconductive heating elements and thermally fixing the regions of saidsealing cover to said microplate; and actively cooling said microplatein a manner that a temperature of said liquid samples is kept below apredefined temperature when heating said sealing cover.
 2. The methodaccording to claim 1, in which said sealing cover is pressed on themicroplate while heating said sealing cover, wherein said pressing isnot stopped before solidification of the sealing cover is reached. 3.The method according to claim 1, wherein said microplate is cooled priorto starting heating said sealing cover and/or after heat-sealing saidmicroplate.
 4. The method according to claim 1, wherein the heating stepcomprises: preheating the heating elements by ramping up the temperatureof the heating elements to a temperature above the melting point of thesealing cover; maintaining the temperature above the melting point ofthe sealing cover at a constant level for a period of time during thepreheating step; and sealing the sealing cover and the microplatetogether by raising the temperature of the heating elements further to apredetermined level.
 5. A method for automatically heat-sealing amicroplate provided with a plurality of open-top wells for receivingliquid samples, wherein said microplate comprises protruding rimssurrounding openings of said wells, said method comprising the followingsteps: disposing a thermally fixable sealing cover over said microplate,wherein said sealing cover comprises regions in opposite relationshipwith respect to said protruding rims; contacting said regions of thesealing cover with a heating device comprising (a) a plurality ofdiscrete electrically conductive heating elements each individuallyconfigured to generate Ohmic heat, and (b) a plurality of non-heatedzones positioned adjacent to the heating elements and in oppositerelationship with respect to the open-top wells, and generating amechanical pressure acting on the sealing cover to press the sealingcover onto the microplate; exclusively heating the plurality of discreteelectrically conductive heating elements and thermally fixing theregions of the sealing cover to said microplate, the heating and fixingstep comprises (i) preheating the heating elements by ramping up thetemperature of the heated elements to a temperature above the meltingpoint of the sealing cover while maintaining the mechanical pressureacting on the sealing cover; (ii) maintaining the temperature above themelting point of the sealing cover at a constant level for a period oftime during the preheating step; and (iii) sealing the sealing cover andthe microplate together by raising the temperature of the heatingelements further to a predetermined level and kept constant whilemaintaining the mechanical pressure acting on the sealing cover; coolingthe heating elements to a temperature below the melting temperature ofthe sealing device while maintaining the mechanical pressure acting onthe sealing cover; and moving the heating device away from the sealingcover and releasing the mechanical pressure acting on the sealing cover.6. The method according to claim 5, further comprising actively coolingsaid microplate in a manner that a temperature of said liquid samples iskept below a predefined temperature when heating said sealing cover.